Gel patterned surfaces

ABSTRACT

Provided is an array including a solid support having a surface, the surface having a plurality of wells, the wells containing a gel material, the wells being separated from each other by interstitial regions on the surface, the interstitial regions segregating the gel material in each of the wells from the gel material in other wells of the plurality; and a library of target nucleic acids in the gel material, wherein the gel material in each of the wells comprises a single species of the target nucleic acids of the library. Methods for making and using the array are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/118,307, filed Aug. 30, 2018, which itself is a divisional of U.S.patent application Ser. No. 15/358,846, filed Nov. 22, 2016 (now U.S.Pat. No. 10,668,444, issued Jun. 2, 2020), which itself is a divisionalof U.S. patent application Ser. No. 13/787,396, filed Mar. 6, 2013 (nowU.S. Pat. No. 9,512,422, issued Dec. 6, 2016), which itself claims thebenefit of U.S. Provisional Application Ser. No. 61/769,289, filed Feb.26, 2013, each of which is incorporated herein by reference in itsentirety.

BACKGROUND

The present disclosure relates generally to solid-phase analyticalchemistry, and has specific applicability to nucleic acid arrays forhigh throughput genomics analysis. The task of cataloguing human geneticvariation and correlating this variation with susceptibility to diseasestands to benefit from advances in genome wide sequencing methodologies.This cataloguing effort holds promise for identifying the markers ineach person's genome that will help medical professionals determinesusceptibility of that person to disease, responsiveness to specifictherapies such as prescription drugs, susceptibility to dangerous drugside effects and other medically actionable characteristics. Thecataloguing effort is well under way. This is due in large part tocommercially available genome sequencing methodologies which aresufficiently cost effective to allow test subjects to be evaluated in aresearch setting. Improvements in sequencing methodologies are needed toaccelerate the cataloguing effort. Moreover, the relatively high cost ofsequencing has hindered the technology from moving beyond the researchcenters and into the clinic where doctors can obtain sequences forpatients in the general population.

Sequencing methodologies and the systems used to carry them out, exploita complex collection of technologies. Improvements in some of thesetechnologies have been shown to provide substantial cost reductions.However, it is difficult to predict which if any is amenable to costreducing improvements. Given the dependencies between the technologiesin the sequencing systems it is even more difficult to predict which canbe modified without having an adverse impact on the overall performanceof the methodology or system. Thus, there exists a need to identifyimprovements that can bring the promise of genomics research to theclinic where lives can be improved and in many cases saved. The presentinvention satisfies this need and provides related advantages as well.

BRIEF SUMMARY

The present disclosure provides an array including a solid supporthaving a surface, the surface having a plurality of wells, the wellscontaining a gel material, the wells being separated from each other byinterstitial regions on the surface, the interstitial regionssegregating the gel material in each of the wells from the gel materialin other wells of the plurality; and a library of target nucleic acidsin the gel material, wherein the gel material in each of the wellscomprises a single species of the target nucleic acids of the library.

In some embodiments the substrate is configured as an array of wells andthe analytes are nucleic acids. Accordingly, this disclosure provides anarray that includes a solid support having a surface, the surface havinga plurality of wells, the wells containing a gel material, the wellsbeing separated from each other by interstitial regions on the surface,the interstitial regions segregating the gel material in each of thewells from the gel material in other wells of the plurality; and alibrary of target nucleic acids in the gel material, wherein the gelmaterial in each of the wells includes a single species of the targetnucleic acids of the library.

The present disclosure also provides a method of making a substrate. Themethod can include the steps of (a) providing a solid support having aplanar surface, wherein the planar surface is interrupted by one or moreconcave features and wherein the one or more concave features arebordered by one or more interstitial regions on the planar surface; (b)coating at least a portion of the solid support with a gel material,wherein the portion includes at least one of the concave features and atleast one of the interstitial regions; and (c) polishing the planarsurface to remove the gel material from the at least one interstitialregions and to maintain the gel material in the at least one concavefeature.

Also provided is a method of making a substrate that includes the stepsof (a) providing a solid support having a planar surface, wherein theplanar surface is interrupted by one or more concave features andwherein the one or more concave features are bordered by one or moreinterstitial regions on the planar surface; (b) coating at least aportion of the solid support with a gel material, wherein the portionincludes at least one of the concave features and at least one of theinterstitial regions, wherein the gel material is bound to a probe orcapable of binding to a probe; and (c) polishing the planar surface toremove or inactivate the gel material at the at least one interstitialregions, whereby the at least one concave feature retains the gelmaterial that is bound to a probe or capable of binding to a probe andwhereby the at least one interstitial regions is not bound to the probeand is not capable of binding to the probe.

A method of making an array can include the steps of (a) providing asolid support having a surface with a plurality of wells, the wellscontaining a gel material, the wells being separated from each other byinterstitial regions on the surface, the interstitial regionssegregating the gel material in each of the wells from the gel materialin other wells of the plurality; (b) delivering a library of targetnucleic acids to the wells of the solid support to produce an array ofwells that have a single species of target nucleic acid attached to thegel material in each well, wherein different wells in the array havedifferent target nucleic acid species from the library; and (c)amplifying the target nucleic acids attached to the gel material in thewells of the array to produce a clonal population of an individualtarget nucleic acid at each of the wells of the array.

This disclosure further provides a method of detecting analytes. Themethod can include the steps of (a) providing a solid support having aplanar surface, wherein the planar surface is interrupted by one or moreconcave features, wherein the concave features contain gel material,wherein the one or more concave features are bordered by one or moreinterstitial regions on the planar surface, the interstitial regionsbeing substantially devoid of the gel material, and wherein the gelmaterial is attached to or contains target analytes; (b) contacting thesolid support with probes under conditions wherein the target analytesinteract specifically with the probes; and (c) detecting the solidsupport to distinguish at least a subset of the target analytes thatinteract with one or more of the probes.

In particular embodiments nucleic acids are the analytes that aredetected and the concave features are wells. For example a method ofdetecting nucleic acids can include the steps of (a) providing a solidsupport having a surface and a library of nucleic acids, the surfacehaving a plurality of wells, the wells containing a gel material, thewells being separated from each other by interstitial regions on thesurface, the interstitial regions segregating the gel material in eachof the wells from the gel material in other wells of the plurality, asingle species of the target nucleic acids of the library being attachedto the gel material in each of the wells; (b) contacting the solidsupport with at least one probe that binds to the target nucleic acids;and (c) detecting the solid support to distinguish the wells having atarget nucleic acid species that binds to the at least one probe.

The compositions, apparatus, and methods of the present disclosure areexemplified herein with regard to gel material. It will be understoodthat the gel material is exemplary and can be replaced with otherorganic materials including, for example, polymers that may form asurface coating and may not necessarily be considered as gels, per se.Methods set forth herein for applying gel material to a surface,removing the gel material from interstitial regions, attaching analytesto the gel material, using the resulting arrays in analytical orpreparative methods etc., can be readily adapted by replacing the gelmaterial with non-gel materials.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a diagrammatic representation of a method for making andusing a patterned array of DNA features, wherein each feature is a wellhaving gel material that is attached to a cluster of DNA and the arrayis used in a sequencing technique.

FIGS. 2A-2C show images from a BeadChip substrate modified to have gelmaterial in the wells instead of beads. FIG. 2A: bright field imagesobtained before polishing. FIGS. 2B-2C: fluorescent images obtainedafter polishing and hybridization to fluorescently labeledoligonucleotides.

FIGS. 3A-3C—FIG. 3A shows a schematic process flow utilizingphotolithography and a Cr hard mask along with reactive ion etching tofabricate concave features in a substrate; FIG. 3B shows example SEMimages of wells and portions of a fiducial in a glass substrate; and inFIG. 3C is an image of a wafer, an image of a portion of the wafer thatincludes a fiducial and an array of wells, and an image from a portionof the array that includes wells.

FIG. 4 shows high resolution fluorescence microscope images of nanowellsubstrates showing patterned gel features on the nanowell substrateafter the substrate is coated with PAZAM and polished with a silica beadslurry. The PAZAM is labeled with a dye for visualization purposes.

FIGS. 5A-5B show a multi-color image merge obtained from a HiSeqsequencing cycle of a 1.5 μm pitch nanowell substrate having patternedclusters. FIG. 5A: image showing a field of clusters patterned ingel-containing wells along with four bulls-eye fiducials. FIG. 5B: ahigher resolution image showing the mixture of colors (due to a mixedpopulation of amplicons) in a single bulls-eye fiducial.

FIGS. 6A-6C show—FIG. 6A: multi-color merge of patterned clusters in aHiseq sequencing run with a 750 nm pitch nanowell substrate; FIG. 6B:nearest neighbor curve showing the array is ordered and clusters arepassing quality filters; and FIG. 6C: sequencing quality metrics showingthat at a density of 1.6 million clusters/mm² quality filters weresuccessfully passed.

FIG. 7 is a plot of Fraction clonal vs. Fraction occupied with the curveexpected for a Poisson distribution, a straight line expected for idealclonality and occupancy and an X for the average measure obtained from asequencing run with a substrate having a pattern of gel-containingnanowells.

DETAILED DESCRIPTION

This disclosure provides structured substrates, methods for makingstructured substrates and methods for using structured substrates. Inparticular embodiments the substrates include a solid support havingconcave regions, such as wells, that contain gel material (e.g. beingcoated by the gel material). The gel material can in turn be attached toan analyte of interest, such as a nucleic acid. In particularembodiments the gel-containing regions are discrete, being separated byinterstitial regions that lack the ability to attach the analyte ofinterest. For example, the interstitial regions may lack the gelmaterial. Alternatively, the gel material in the interstitial regionsmay be inactivated or otherwise modified to lack an activity orcharacteristic of the gel material in the concave regions, such as theability to support analyte attachment. The resulting segregation of thegel regions provides advantages when carrying out reactions on theanalytes and/or detecting the analytes. An exemplary advantage can bedemonstrated in the instance of an array of target nucleic acidsdistributed among gel-containing wells. Here, an amplification reactioncan be carried out on the structured substrate using the nucleic acidsas templates to form nucleic acid colonies that grow in or on the gel(e.g. nucleic acid features of the array). The interstitial regionsfunction to confine the area of growth for the colony. The individualfeatures of the resulting array can be distinguished with relative easedue to the discrete pattern created by the gel-containing wells. Thepattern can also provide benefits of increasing the density of featuresand reducing processing requirements for image registration as comparedto random arrays of nucleic acids.

An exemplary process for making a patterned array of nucleic acids isshown in FIG. 1. A profile view of a well-patterned substrate is showndiagrammatically. The wells have a pitch (center-to-center spacing) of1.5 μm and the diameter of each well is 0.5 μm in the example. The wellpatterned substrate can be coated with a gel material such that materialenters the wells and coats the interstitial regions. The resultinggel-coated substrate can be polished to remove gel material from theinterstitial regions, leaving gel material in the wells, thereby forminga gel-patterned substrate. The gel can function to support capture of aDNA template and amplification of the template. For example, the gel canbe grafted with oligonucleotide primers prior to surface coating, aftersurface coating and before polishing, or after polishing. The primerscan function to capture the DNA templates and to prime amplificationusing the captured templates. The resulting DNA-patterned substrate canbe analyzed for example in a sequencing technique.

A patterned array of nucleic acids in gel-containing wells providesmultiple advantages for DNA sequencing. Examples of advantages comparedto random arrays (i.e. arrays having a random pattern of features)include increased density of feature packing, increased control andtuning of feature density using concentration-independent templateseeding, reduced processing requirements for image registration andincreased ease of signal extraction. A further advantage can be derivedfrom spatial confinement of nucleic acid populations provided by eachfeature. A feature of a patterned array of the present disclosure canfunction to restrict the area or volume within which a nucleic acidcolony will grow (for example, via cluster amplification). Absent areaor volume restrictions, some nucleic acid colonies may amplify to alarger size than others due to differences in percent content of guanineand cytosine in their sequences (i.e. GC content) which influencerelative amplification rates. For the methods and compositions set forthherein, the volume or area of individual features can be selected toprevent or minimize differences in nucleic acid colony sizes that wouldotherwise occur from an amplification reaction due to differences in GCcontent between the template species being amplified. For example, thevolume or area of the features can be sufficiently small to cap growthof the fastest growing colonies while allowing slower growing coloniesto effectively fill the feature upon completion of the amplificationreaction.

In particular embodiments, this disclosure provides fabrication of wells(e.g. microwells or nanowells) on glass, silicon, plastic or othersuitable solid supports with patterned, covalently-linked gel such aspoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM, see,for example, U.S. Prov. Pat. App. Ser. No. 61/753,833, which isincorporated herein by reference). The process creates gel pads used forsequencing that can be stable over sequencing runs with a large numberof cycles. The covalent linking of the polymer to the wells is helpfulfor maintaining the gel in the structured features throughout thelifetime of the structured substrate during a variety of uses. Howeverin many embodiments, the gel need not be covalently linked to the wells.For example, in some conditions silane free acrylamide (SFA, see, forexample, US Pat. App. Pub. No. 2011/0059865 A1, which is incorporatedherein by reference) which is not covalently attached to any part of thestructured substrate, can be used as the gel material.

In particular embodiments, a structured substrate can be made bypatterning a solid support material with wells (e.g. microwells ornanowells), coating the patterned support with a gel material (e.g.PAZAM, SFA or chemically modified variants thereof, such as theazidolyzed version of SFA (azido-SFA)) and polishing the gel coatedsupport, for example via chemical or mechanical polishing, therebyretaining gel in the wells but removing or inactivating substantiallyall of the gel from the interstitial regions on the surface of thestructured substrate between the wells. Primer nucleic acids can beattached to gel material. A solution of target nucleic acids (e.g. afragmented human genome) can then be contacted with the polishedsubstrate such that individual target nucleic acids will seed individualwells via interactions with primers attached to the gel material;however, the target nucleic acids will not occupy the interstitialregions due to absence or inactivity of the gel material. Amplificationof the target nucleic acids will be confined to the wells since absenceor inactivity of gel in the interstitial regions prevents outwardmigration of the growing nucleic acid colony. The process isconveniently manufacturable, being scalable and utilizing conventionalmicro- or nano-fabrication methods.

In particular embodiments, fiducial markers are included on a structuredsubstrate to facilitate identification and localization of individualfeatures (e.g. wells or other gel-containing concave features). Fiducialmarkers are particularly useful for structured substrates having aspatially ordered pattern of features since the fiducial markers providea point of reference for relative locations of other features. Fiducialmarkers can be used for registering images of random arrays as well, butthe inherent disorder of clusters can be used instead, for example, asused with random arrays generated on commercial sequence platforms suchas the HiSeq, Genome Analyzer or MiSeq platforms from Illumina, Inc.(San Diego, Calif.). Fiducial markers are especially beneficial forapplications where the structured substrate is detected repeatedly tofollow changes occurring at individual features over time. Fiducialmarkers allow individual nucleic acid clusters to be followed throughsequential images obtained over multiple sequencing cycles, such thatthe sequence of individual clusters can be discretely determined.

This disclosure provides a fiducial marker having a pattern of concaveregion(s) and interstitial region(s). An exemplary design for a fiducialmarker is a set of concentric circles having an alternating pattern oftwo or more of the following: a concave ring, interstitial ring and aring of wells or other concave features (e.g. a “bulls-eye”). In someembodiments concave region(s) of a fiducial marker contain gel material,whereas interstitial regions do not. This differential location of thegel on the surface can be achieved using the gel coating and polishingmethods set forth herein. Typically a detection method is used that candistinguish gel-containing regions from interstitial regions. In somecases, the distinction can be based on the presence of a particularanalyte in the gel regions that is absent from the interstitial regions.For example, in the case of nucleic acid arrays, the gel-containingregion of a fiducial marker can contain nucleic acids that are labeledvia the same methods that are used to label target nucleic acids on thearray. Thus, fiducial markers can be conveniently fabricated using thesame methods used to fabricate analyte features. Accordingly, ifdesired, fiducial markers and analyte features can be fabricatedsimultaneously across one or more steps. Another useful fiducial markerthat can be used in the structured substrates and methods set forthherein is one having sub-regions where the pattern of wells (or otherconcave features) in one subregion is rotated with respect to thepattern in another subregion. Such fiducial grids can be configured andused for image registration as set forth in U.S. Ser. No. 13/267,565,which is incorporated herein by reference.

As a further example, beads can be used as a fiducial. The beads caninclude a label such as a fluorophore. In this case, a surface can haveat least two types of wells (or other concave features). Relativelylarge wells can accommodate one or more fiducial beads, whereas smallerwells, being too small to contain a bead, will only have gel material.Thus, the smaller wells function as analytical features for analysis andthe larger, bead-filled wells function as fiducials. As an alternativeto wells the fiducial features can be channels, such as those present inthe bulls-eye configuration exemplified above, and the channels can havedimensions that accommodate the beads. As such, several beads can beplaced in the channel to create a fiducial, for example, in the apparentshape of a string of beads.

Patterned arrays, methods for their manufacture and methods for theiruse are exemplified herein with regard to a gel material that is used toattach analytes of interest. It will be understood that the gel materialis exemplary and can be replaced with other organic materials that canbe used to mediate localization of analytes to features on a surface.Such organic materials include for example, polymers that may form asurface coating and may not necessarily be considered as gels, per se. Aspecific example is a polymer formed by ATRP (atom transfer radicalpolymerization) or surface initiated polymerization processes. Methodsset forth herein for applying gel material to a surface, removing thegel material from interstitial regions, using the resulting arrays inanalytical or preparative methods etc., can be readily adapted for usewith non-gel materials.

Terms used herein will be understood to take on their ordinary meaningin the relevant art unless specified otherwise. Several terms usedherein and their meanings are set forth below.

As used herein, the term “attached” refers to the state of two thingsbeing joined, fastened, adhered, connected or bound to each other. Forexample, an analyte, such as a nucleic acid, can be attached to amaterial, such as a gel or solid support, by a covalent or non-covalentbond. A covalent bond is characterized by the sharing of pairs ofelectrons between atoms. A non-covalent bond is a chemical bond thatdoes not involve the sharing of pairs of electrons and can include, forexample, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilicinteractions and hydrophobic interactions.

As used herein, the term “clonal population” refers to a population ofnucleic acids that is homogeneous with respect to a particularnucleotide sequence. The homogenous sequence is typically at least 10nucleotides long, but can be even longer including for example, at least50, 100, 250, 500, 1000 or 2500 nucleotides long. A clonal populationcan be derived from a single target nucleic acid or template nucleicacid. A clonal population can include at least 2, 5, 10, 100, 1000 ormore copies of a target nucleotide sequence. The copies can be presentin a single nucleic acid molecule, for example, as a concatamer or thecopies can be present on separate nucleic acid molecules (i.e. a clonalpopulation can include at least 2, 5, 10, 100, 1000 or more nucleic acidmolecules having the same target nucleotide sequence). Typically, all ofthe nucleic acids in a clonal population will have the same nucleotidesequence. It will be understood that a negligible number of contaminantnucleic acids or mutations (e.g. due to amplification artifacts) canoccur in a clonal population without departing from clonality. Thus, apopulation can be at least 80%, 90%, 95% or 99% clonal. In some cases100% pure clonal populations may be present.

As used herein, the term “coat,” when used as a verb, is intended tomean providing a layer or covering on a surface. At least a portion ofthe surface can be provided with a layer or cover. In some cases theentire surface can be provided with a layer or cover. In alternativecases only a portion of the surface will be provided with a layer orcovering. The term “coat,” when used to describe the relationshipbetween a surface and a material, is intended to mean that the materialis present as a layer or cover on the surface. The material can seal thesurface, for example, preventing contact of liquid or gas with thesurface. However, the material need not form a seal. For example, thematerial can be porous to liquid, gas, or one or more components carriedin a liquid or gas. Exemplary materials that can coat a surface include,but are not limited to, a gel, polymer, organic polymer, liquid, metal,a second surface, plastic, silica, or gas.

As used herein, the term “concave feature,” when used in reference to asolid support, refers to a recess or indentation in the solid support.Exemplary concave features include, but are not limited to, a well, pit,hole, depression, channel, or trough. A concave feature can optionallyhave a curved cross section (in the dimension orthogonal to the surfaceof the solid support); however, a cross section with one or more linearsections, angles or corners is also possible. Cross sections withcombinations of curved and linear sections are also possible. Generally,a concave feature need not pass completely through the solid support,for example, instead having a bottom surface or point in the substrate.

As used herein, the term “different”, when used in reference to nucleicacids, means that the nucleic acids have nucleotide sequences that arenot the same as each other. Two or more nucleic acids can havenucleotide sequences that are different along their entire length.Alternatively, two or more nucleic acids can have nucleotide sequencesthat are different along a substantial portion of their length. Forexample, two or more nucleic acids can have target nucleotide sequenceportions that are different for the two or more molecules while alsohaving a universal sequence portion that is the same on the two or moremolecules.

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection.Exceptions can occur if explicit disclosure or context clearly dictatesotherwise.

As used herein, the term “fluidic access,” when used in reference to amolecule in a fluid and a site in contact with the fluid, refers to theability of the molecule to move in or through the fluid to contact orenter the site. The term can also refer to the ability of the moleculeto separate from or exit the site to enter the solution. Fluidic accesscan occur when there are no barriers that prevent the molecule fromentering the site, contacting the site, separating from the site and/orexiting the site. However, fluidic access is understood to exist even ifdiffusion is retarded, reduced or altered so long as access is notabsolutely prevented.

As used herein, the term “gel material” is intended to mean a semi-rigidmaterial that is permeable to liquids and gases. Typically, gel materialcan swell when liquid is taken up and can contract when liquid isremoved by drying. Exemplary gels include, but are not limited to thosehaving a colloidal structure, such as agarose; polymer mesh structure,such as gelatin; or cross-linked polymer structure, such aspolyacrylamide, SFA (see, for example, US Pat. App. Pub. No.2011/0059865 A1, which is incorporated herein by reference) or PAZAM(see, for example, U.S. Prov. Pat. App. Ser. No. 61/753,833, which isincorporated herein by reference). Particularly useful gel material willconform to the shape of a well or other concave feature where itresides. Some useful gel materials can both (a) conform to the shape ofthe well or other concave feature where it resides and (b) have a volumethat does not substantially exceed the volume of the well or concavefeature where it resides.

As used herein, the term “interstitial region” refers to an area in asubstrate or on a surface that separates other areas of the substrate orsurface. For example, an interstitial region can separate one concavefeature of an array from another concave feature of the array. The tworegions that are separated from each other can be discrete, lackingcontact with each other. In another example, an interstitial region canseparate a first portion of a feature from a second portion of afeature. In many embodiments the interstitial region is continuouswhereas the features are discrete, for example, as is the case for anarray of wells in an otherwise continuous surface. The separationprovided by an interstitial region can be partial or full separation.Interstitial regions will typically have a surface material that differsfrom the surface material of the features on the surface. For example,features of an array can have an amount or concentration of gel materialor analytes that exceeds the amount or concentration present at theinterstitial regions. In some embodiments the gel material or analytesmay not be present at the interstitial regions.

As used herein, the term “library,” when used in reference to analytes,refers to a collection of analytes having different chemicalcompositions. Typically, the analytes in a library will be differentspecies having a common feature or characteristic of a genera or class,but otherwise differing in some way. For example, a library can includenucleic acid species that differ in nucleotide sequence, but that aresimilar with respect to having a sugar-phosphate backbone.

As used herein, the terms “nucleic acid” and “nucleotide” are intendedto be consistent with their use in the art and to include naturallyoccurring species or functional analogs thereof. Particularly usefulfunctional analogs of nucleic acids are capable of hybridizing to anucleic acid in a sequence specific fashion or capable of being used asa template for replication of a particular nucleotide sequence.Naturally occurring nucleic acids generally have a backbone containingphosphodiester bonds. An analog structure can have an alternate backbonelinkage including any of a variety of those known in the art. Naturallyoccurring nucleic acids generally have a deoxyribose sugar (e.g. foundin deoxyribonucleic acid (DNA)) or a ribose sugar (e.g. found inribonucleic acid (RNA)). A nucleic acid can contain nucleotides havingany of a variety of analogs of these sugar moieties that are known inthe art. A nucleic acid can include native or non-native nucleotides. Inthis regard, a native deoxyribonucleic acid can have one or more basesselected from the group consisting of adenine, thymine, cytosine orguanine and a ribonucleic acid can have one or more bases selected fromthe group consisting of uracil, adenine, cytosine or guanine. Usefulnon-native bases that can be included in a nucleic acid or nucleotideare known in the art. The terms “probe” or “target,” when used inreference to a nucleic acid, are intended as semantic identifiers forthe nucleic acid in the context of a method or composition set forthherein and does not necessarily limit the structure or function of thenucleic acid beyond what is otherwise explicitly indicated. The terms“probe” and “target” can be similarly applied to other analytes such asproteins, small molecules, cells or the like.

As used herein, the term “random pattern,” when used in reference towells on a surface, means that the relative locations of a subset ofwells in one region of the surface is not known or predictable from thelocations of a subset of wells in another region of the surface. Thesubset used for the measure will generally include at least 3 wells butcan include at least, 4, 5, 6, 10 or more wells. A random patterngenerally does not include multiple repetitions of any sub-patterns. Theterm can be applied to other concave features besides wells.

As used herein, the term “repeating pattern,” when used in reference towells on a surface, means that the relative locations of a subset ofwells in one region of the surface is the same as the relative locationsof a subset of wells in at least one other region of the surface. Thus,the relative locations for wells in one region of a repeating patternare generally predictable from the relative locations of wells inanother region of the repeating pattern. The subset used for the measurewill generally include at least 3 wells but can include at least, 4, 5,6, 10 or more wells. Exemplary repeating patterns include rectilinearpatterns and hexagonal patterns. A repeating pattern can includemultiple repetitions of a sub-pattern. The term can be applied to otherconcave features besides wells.

As used herein, the term “segregate,” when used in reference to gelmaterial in two wells (or at two other features), means to separate orisolate the gel material in one of the wells (or at one of the features)from the gel material in the other well (or at the other feature). Thus,the gel material in the first well (or at the first feature) is not indirect contact with the gel material in the other well (or at the otherfeature). In some embodiments, the gel material in the two wells (or atthe two features) is in indirect contact, for example, via a solutionthat contacts the two wells (or features). Alternatively, the gelmaterial in the two wells (or at the two features) is not even inindirect contact. An interstitial region on a surface can segregate thegel material in two wells (or at two features) by being devoid of thegel material. In particular embodiments, a gel material can bediscontinuous on a surface, being present at concave features, such aswells, but not present at interstitial regions between the features.

As used herein, the term “surface” is intended to mean an external partor external layer of a solid support or gel material. The surface can bein contact with another material such as a gas, liquid, gel, polymer,organic polymer, second surface of a similar or different material,metal, or coat. The surface, or regions thereof, can be substantiallyflat. The surface can have surface features such as wells, pits,channels, ridges, raised regions, pegs, posts or the like.

As used herein, the term “single species” means substantially one andonly one species of a particular genera. The term is not necessarilyintended to limit the number of representatives of a single species thatare present. For example, a population of nucleic acid molecules eachhaving the same nucleotide sequence comprise a single species of nucleicacid. The term “single” in this context is not intended to exclude thepresence of other things that are not within the relevant genera. Forexample, a well that contains a single species of target nucleic acidfrom a library can include multiple nucleic acids having the samesequence, will exclude other target nucleic from the library, but neednot necessarily exclude any other non-nucleic acid components. It willbe understood that an apparent single species population can have asmall amount of another species present at a level that is considered bythose skilled in the art to be a negligible level of contamination orartifact for the particular use of the population. For example, anucleic acid cluster, derived from a single template having a firstsequence, will be considered to have an apparent single species if theamount of any nucleic acid molecules having a second sequence issufficiently low to be undetectable or ignored when the first sequenceis detected. Alternatively, an absolute single species population willhave one and only one species.

As used herein, the term “solid support” refers to a rigid substratethat is insoluble in aqueous liquid. The substrate can be non-porous orporous. The substrate can optionally be capable of taking up a liquid(e.g. due to porosity) but will typically be sufficiently rigid that thesubstrate does not swell substantially when taking up the liquid anddoes not contract substantially when the liquid is removed by drying. Anonporous solid support is generally impermeable to liquids or gases.Solid supports can optionally be inert to a chemistry that is used tomodify a gel. For example, a solid support can be inert to chemistryused to attach analytes, such as nucleic acids, to gels in a method setforth herein. Exemplary solid supports include, but are not limited to,glass and modified or functionalized glass, plastics (includingacrylics, polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon®,cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor,silica or silica-based materials including silicon and modified silicon,carbon, metals, inorganic glasses, optical fiber bundles, and polymers.Particularly useful solid supports for some embodiments are locatedwithin a flow cell apparatus. Exemplary flow cells are set forth infurther detail below.

As used herein, the term “well” refers to a discrete concave feature ina solid support having a surface opening that is completely surroundedby interstitial region(s) of the surface. Wells can have any of avariety of shapes at their opening in a surface including but notlimited to round, elliptical, square, polygonal, star shaped (with anynumber of vertices) etc. The cross section of a well taken orthogonallywith the surface can be curved, square, polygonal, hyperbolic, conical,angular, etc.

The embodiments set forth below and recited in the claims can beunderstood in view of the above definitions.

The present disclosure provides a substrate that includes a solidsupport having a surface, the surface having at least one concavefeature, the at least one concave feature containing a gel material, theat least one concave feature being bordered by at least one interstitialregion on the surface; and a library of analytes in the gel material,wherein the gel material in each of the wells includes a single speciesof the analytes of the library.

In some embodiments the substrate is configured as an array of wells andthe analytes are nucleic acids. Accordingly, this disclosure provides anarray that includes a solid support having a surface, the surface havinga plurality of wells, the wells containing a gel material, the wellsbeing separated from each other by interstitial regions on the surface,the interstitial regions segregating the gel material in each of thewells from the gel material in other wells of the plurality; and alibrary of target nucleic acids in the gel material, wherein the gelmaterial in each of the wells includes a single species of the targetnucleic acids of the library.

A solid support used in a structured substrate set forth herein can bemade from any of a variety of materials set forth herein, for example,above in the definitions, below in the examples or immediatelyfollowing. A particularly useful material is glass. Other suitablesubstrate materials may include polymeric materials, plastics, silicon,quartz (fused silica), borofloat glass, silica, silica-based materials,carbon, metals, an optical fiber or optical fiber bundles, sapphire, orplastic materials such as COCs and epoxies. The particular material canbe selected based on properties desired for a particular use. Forexample, materials that are transparent to a desired wavelength ofradiation are useful for analytical techniques that will utilizeradiation of the desired wavelength, such as one or more of thetechniques set forth herein. Conversely, it may be desirable to select amaterial that does not pass radiation of a certain wavelength (e.g.being opaque, absorptive or reflective). This can be useful forformation of a mask to be used during manufacture of the structuredsubstrate, such as a method set forth herein; or to be used for achemical reaction or analytical detection carried out using thestructured substrate, such as those set forth herein. Other propertiesof a material that can be exploited are inertness or reactivity tocertain reagents used in a downstream process, such as those set forthherein; or ease of manipulation or low cost during a manufacturingprocess manufacture, such as those set forth herein. Further examples ofmaterials that can be used in the structured substrates or methods ofthe present disclosure are described in U.S. Ser. No. 13/661,524 and USPat. App. Pub. No. 2012/0316086 A1, each of which is incorporated hereinby reference.

In a particular embodiment, a Sol-Gel based substrate can be made andused. Sol-Gel based patterning can be accomplished by coating a rigid orflexible substrate, such as glass, silicon, plastic, metal or the like,with a Sol-Gel coating can be carried out, for example, through spincoating, dipping or spray coating. The Sol-Gel can be provided in aliquid state when applied to the substrate and can contain either photo-or thermal-initiators that enable curing (making the liquid a gel)through the exposure of the Sol-Gel to either light or heat. Subsequentto coating the substrate with the Sol-Gel, and prior to curing thematerial, the Sol-Gel can be imprinted with a template (threedimensional stamp) that has either a single or plurality of protrudingfeature(s). The template can be made, for example, of silicon, glass(such as quartz), metal (such as nickel), plastic or polymer (such asPDMS). Imprinting of the stamp into the Sol-Gel can be accomplished byplacing the template in contact with the Sol-Gel. When the template isin contact with the Sol-Gel, the Sol-Gel redistributes to conformallysurround the structure of the template. When the template is in contactwith the Sol-Gel, the redistribution of the Sol-Gel can be driven eitherthrough an external force applied to the template or the substrate, orthrough capillary forces intrinsic to the nature of the patternedtemplate. When the template is in contact with the Sol-Gel, the stack ofsubstrate+Sol-Gel+template can be exposed to light or heat to cure theSol-Gel and to lock the pattern originally in the template into theSol-Gel. This patterning process is conventionally referred to asnanoimprint lithography. Following curing of the Sol-Gel, the templatecan be separated from the substrate+Sol-Gel stack, and the template canbe either discarded or re-used to pattern another substrate coated withun-cured Sol-Gel. The substrate with the cured, patterned Sol-Gel canthen be taken into a chemical deposition process, or if a pure glasslike surface is desired, the substrate+patterned Sol-Gel stack can betaken through a thermal process (sintering) that will remove organicmaterial that was originally present in the Sol-Gel. This is notrequired but can make the substrate a pure SiO₂ material that hasadvantages in certain chemical attachment schemes.

Another approach to producing a patterned substrate is to use a plasticmaterial such as COC or COP (such as Zeonor or Topas) and perform athermal embossing process to create the array of indentations. Theprocess is similar to nanoimprint lithography. A substrate of plasticcan be mounted on a chuck that has the ability to be temperaturecontrolled. The plastic substrate can then be heated to a temperaturesuch that the outer skin of the plastic exceeds the glass transitiontemperature. While the substrate is at the elevated temperature, thetemplate is put into hard contact with a template (e.g. quartz, silicon,polymeric or metallic). The template typically has an external forceapplied to ensure that the plastic completely conformally coats thestructured template. While in contact at the elevated temperature, theplastic redistributes itself to become a negative replica of thetemplate; for example if the template has an array of posts then theembossed plastic results in an array of wells. While the template is incontact with the plastic substrate, the temperature of the substrate isreduced thus locking the embossed pattern into the substrate.

A concave feature that is on a substrate can have any of a variety ofshapes. In terms of the shape on the surface, the feature can havecurved sides, linear sides, corners or a combination thereof. Forexample, the features can be wells having openings in the surface thatare circular, oval, square, polygonal, star-shaped (with any number ofvertices), or irregularly shaped. The features can be channels and theshape of the channels on the surface can include sides that are curved,linear, angled or a combination thereof. Other channel features can belinear, serpentine, rectangular, square, triangular, circular, oval,hyperbolic, or a combination thereof. The channels can have one or morebranches or corners. The channels can connect two points on a surface,one or both of which can be the edge of the substrate. FIG. 3B and FIG.3C shows exemplary channel features in bulls-eye fiducials along withwells within and surrounding the bulls-eye fiducials.

The cross sectional shape of a concave feature, taken orthogonal to thesurface, can have walls that are curved, linear or a combination of thetwo. Thus, the cross-sectional shape can be a portion of a circle oroval (e.g. U-shaped), or can have two or more linear sides that meet atcorners (e.g. V-, square-, polygonal- or star-shaped). In terms ofcross-sectional shape, the bottom of the concave feature can benarrower, wider, or roughly the same as the opening on the surface.These cross sectional shapes can be illustrated for the case where theconcave feature is a well, in which case, the opening in the surfacewill be roughly the same area as the bottom of the well when the wellhas a cylindrical cross section, whereas the bottom of the well willhave a different area (typically smaller) than the area at the openingon the surface when the well has a conical cross section. Of course, thecross sections, although illustrated for wells, can apply to channelsalso.

For embodiments where the concave features form wells, each well canhave any volume that is capable of confining a liquid. The minimum ormaximum volume can be selected, for example, to accommodate thethroughput (e.g. multiplexity), resolution, analyte composition, oranalyte reactivity expected for downstream uses of the substrate. Forexample, the volume can be at least 1×10⁻³ μm³, 1×10⁻² μm³, 0.1 μm³, 1μm³, 10 μm³, 100 μm³ or more. Alternatively or additionally, the volumecan be at most 1×10⁴ μm³, 1×10³ μm³, 100 μm³, 10 μm³, 1 μm³, 0.1 μm³ orless. It will be understood that gel material can fill all or part ofthe volume of a well. The volume of gel in an individual well can begreater than, less than or between the values specified above.

The area occupied by each well opening on a surface can be selectedbased upon similar criteria as those set forth above for well volume.For example, the area for each well opening on a surface can be at least1×10⁻³ μm², 1×10⁻² μm², 0.1 μm², 1 μm², 10 μm², 100 μm² or more.Alternatively or additionally, the area can be at most 1×10³ μm², 100μm², 10 μm², 1 μm², 0.1 μm², 1×10⁻² μm², or less. The depth of each wellcan be at least 0.1 μm, 1 μm, 10 μm, 100 μm or more. Alternatively oradditionally, the depth can be at most 1×10³ μm, 100 μm, 10 μm, 1 μm,0.1 μm or less.

Many different layouts of wells or other concave features may beenvisaged, including regular, repeating, and non-regular patterns. Forexample, wells can be disposed in a hexagonal grid for close packing andimproved density. Other layouts may include, for example, rectilinear(i.e., rectangular) layouts, triangular layouts, and so forth. Theparticular layouts, and differences between the layouts of differentdomains, if used, may follow the teachings of U.S. Pat. No. 7,813,013,and/or of U.S. patent application Ser. No. 13/267,565, each of which ishereby incorporated by reference. Any of a variety of crystalline orpoly-crystalline patterns can be useful.

A pattern of wells can be characterized in terms of the average pitch(i.e. center-to-center spacing) for the wells. Again, the pattern can beregular such that the coefficient of variation around the average pitchis small or the pattern can be non-regular in which case the coefficientof variation can be relatively large. In either case, the average pitchcan be, for example, at least 10 nm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm,100 μm or more. Alternatively or additionally, the average pitch can be,for example, at most 100 μm, 10 μm, 5 μm, 1 μm, 0.5 μm 0.1 μm or less.Of course, the average pitch for a particular pattern of wells can bebetween one of the lower values and one of the upper values selectedfrom the ranges above.

A pattern of wells can also be characterized with respect to the densityof wells (i.e. number of wells) in a defined area. For example, thewells may be present at a density of approximately 2 million per mm². Inaccordance with the manufacturing methods set forth herein, the densitycan easily be tuned to different densities including, for example, adensity of at least 100 per mm², 1,000 per mm², 0.1 million per mm², 1million per mm², 2 million per mm², 5 million per mm², 10 million permm², 50 million per mm² or more. Alternatively or additionally, thedensity can be tuned to be no more than 50 million per mm², 10 millionper mm², 5 million per mm², 2 million per mm², 1 million per mm², 0.1million per mm², 1,000 per mm², 100 per mm² or less. Of course, thedensity of wells on a substrate can be between one of the lower valuesand one of the upper values selected from the ranges above.

In particular embodiments, a gel material is used. In some cases, agel-forming (e.g. polymerizable) material is provided to a solid supportin a liquid state and subsequently converted to a gel. Examples ofpolymerizable materials include, without limitation, acrylamide,methacrylamide, hydroxyethyl methacrylate, N-vinyl pyrolidinone orderivatives thereof. Such materials are useful for preparing hydrogels.In some embodiments, the polymerizable material can include two or moredifferent species of compound that form a co-polymer. For example, twoor more different species of acrylamide, methacrylamide, hydroxyethylmethacrylate, N-vinyl pyrolidinone or derivatives thereof can functionas co-monomers that polymerize to form a copolymer hydrogel. Usefulhydrogels include, but are not limited to, silane-free acrylamide (SFA)polymer (see US Pat. App. Pub. No. 2011/0059865 A1, which isincorporated herein by reference),poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM, seeU.S. Prov. Pat. App. Ser. No. 61/753,833, which is incorporated hereinby reference), polyacrylamide polymers formed from acrylamide and anacrylic acid or an acrylic acid containing a vinyl group as described,for example, in WO 00/31148 (incorporated herein by reference);polyacrylamide polymers formed from monomers that form [2+2]photo-cycloaddition reactions, for example, as described in WO 01/01143or WO 03/014392 (each of which is incorporated herein by reference); orpolyacrylamide copolymers described in U.S. Pat. No. 6,465,178, WO01/62982 or WO 00/53812 (each of which is incorporated herein byreference). Chemically treated variants of these gel materials are alsouseful, such as chemically treated SFA made to react witholigonucleotides having a corresponding reactive group (such as theazidolysis of SFA to produce azido-SFA which is reactive with a 5′- or3′-alkynyl modified oligonucleotides) Exemplary hydrogels andpolymerizable materials that can be used to form hydrogels aredescribed, for example, in U.S. Ser. No. 61/753,833 or US Pat. App. Pub.No. 2011/0059865 A1, each of which is incorporated herein by reference.Other useful gels are those that are formed by a temperature dependentchange in state from liquid to gelatinous. Examples include, but are notlimited to agar, agarose, or gelatin.

The gel material that is in a well or other concave feature on thesurface of a structured substrate can be covalently attached to thesurface. For example, PAZAM can be covalently attached to a surfaceusing surface materials and other reagents set forth in U.S. Ser. No.61/753,833, which is incorporated herein by reference, and as set forthin the Examples section herein. However, the gel material need not becovalently attached to wells or other concave features as exemplifiedfor SFA in the Examples section below.

One or more analytes can be present in or on the gel material that ispresent on a structured substrate. The gel-containing substrates of thepresent disclosure are particularly useful for detection of analytes, orfor carrying out synthetic reactions with analytes. Thus, any of avariety of analytes that are to be detected, characterized, modified,synthesized, or the like can be present in or on gel material of asubstrate set forth herein. Exemplary analytes include, but are notlimited to, nucleic acids (e.g. DNA, RNA or analogs thereof), proteins,polysaccharides, cells, antibodies, epitopes, receptors, ligands,enzymes (e.g. kinases, phosphatases or polymerases), small molecule drugcandidates, or the like. A structured substrate can include multipledifferent species from a library of analytes. For example, the speciescan be different antibodies from an antibody library, nucleic acidshaving different sequences from a library of nucleic acids, proteinshaving different structure and/or function from a library of proteins,drug candidates from a combinatorial library of small molecules etc.

In some embodiments, analytes can be distributed on a structuredsubstrate such that they are individually resolvable. For example, asingle molecule of each analyte can be present in each gel-containingwell of a structured substrate. Alternatively, analytes can be presentas colonies or populations such that individual molecules are notnecessarily resolved. The colonies or populations can be homogenous withrespect to containing only a single species of analyte (albeit inmultiple copies). Taking nucleic acids as an example, each well on astructured substrate can include a colony or population of nucleic acidsand every nucleic acid in the colony or population can have the samenucleotide sequence (either single stranded or double stranded). Suchcolonies can be created by cluster amplification or bridge amplificationas set forth in further detail elsewhere herein. Multiple repeats of atarget sequence can be present in a single nucleic acid molecule, suchas a concatamer created using a rolling circle amplification procedure.Thus, the gel material in each well on a structured substrate cancontain multiple copies of a single species of an analyte.Alternatively, a colony or population of analytes that are in a well caninclude two or more different species. For example, one or more wells ona structured substrate can each contain a mixed colony having two ormore different nucleic acid species (i.e. nucleic acid molecules withdifferent sequences). The two or more nucleic acid species in a mixedcolony can be present in non-negligible amounts, for example, allowingmore than one nucleic acid to be detected in the mixed colony.

Analytes can be attached to a gel material. The attachment can becovalent or non-covalent. Exemplary methods and reactants for attachingnucleic acids to gels are described, for example, in US Pat. App. Pub.No. 2011/0059865 A1, or U.S. Prov. Pat. App. Ser. No. 61/753,833, eachof which is incorporated herein by reference. The analytes can benucleic acids and the nucleic acids can be attached to the gel via their3′ oxygen, 5′ oxygen, or at other locations along their length such asvia a base moiety of the 3′ terminal nucleotide, a base moiety of the 5′nucleotide, and/or one or more base moieties elsewhere in the molecule.Non-covalent modes of attachment include, for example, ionicinteractions between nucleic acid and gel, entrapment of nucleic acidwithin pores of a gel, protein-protein interactions, binding betweenreceptors and ligands on the gel and/or nucleic acid, and other knownmodes.

In some embodiments a gel coating that is applied to a surface containsone or more analytes prior to removal of gel material from interstitialregions. Thus, gel material can be present at the interstitial regionsand the gel material at the interstitial regions can be attached to oneor more different analytes. Alternatively, analytes are added to a gelmaterial in concave features after removing gel material from theinterstitial regions.

A structured substrate of the present disclosure can occur in a flowcell. Exemplary flow cells, methods for their manufacture and methodsfor their use are described in US Pat. App. Publ. Nos. 2010/0111768 A1or 2012-0270305 A1; or WO 05/065814, each of which is incorporatedherein by reference. Flow cells provide a convenient format for housingan array that is produced by the methods of the present disclosure andthat is subjected to a sequencing-by-synthesis (SBS) or other techniquethat involves repeated delivery of reagents in cycles (e.g. synthesistechniques or detection techniques having repetitive or cyclic steps).Exemplary detection methods are set forth in further detail below.

In some embodiments a flow-cell or other vessel having multiple surfacesis used. Vessels having multiple surfaces can be used such that only asingle surface has gel-containing concave features (e.g. wells).Alternatively two or more surfaces present in the vessel can havegel-containing concave features. One or more surfaces of a flow cell canbe selectively detected. For example, opposing surfaces in the interiorof a flow cell can be selectively addressed with focused radiation usingmethods known in the art such as confocal techniques. Useful confocaltechniques and devices for selectively directing radiation to multiplesurfaces of a vessel (e.g. a flow cell) are described, for example, inUS Pat. App. Pub. No. 2009/0272914 A1 or U.S. Pat. No. 8,039,817, eachof which is incorporated herein by reference.

The present disclosure provides a method of making a substrate. Themethod can include the steps of (a) providing a solid support having aplanar surface, wherein the planar surface is interrupted by one or moreconcave features and wherein the one or more concave features arebordered by one or more interstitial regions on the planar surface; (b)coating at least a portion of the solid support with a gel material,wherein the portion includes at least one of the concave features and atleast one of the interstitial regions; and (c) polishing the planarsurface to remove the gel material from the at least one interstitialregions and to maintain the gel material in the at least one concavefeature.

A substrate can be fabricated to have concave features using any of avariety of techniques known in the art. In many embodiments, the concavefeatures will be small, on the order of nanometer or micrometerdimensions. In such cases nanofabrication or microfabrication techniquescan be used. Examples of these techniques are set forth elsewhere hereinsuch as in Example II below. Further exemplary nanofabrication andmicrofabrication techniques are described in U.S. Ser. No. 13/661,524and US Pat. App. Publ. No. 2012/0316086 A1, each of which isincorporated herein by reference.

One or more concave features, such as wells, can be coated withpreformed gel material or with a liquid that subsequently forms a gelmaterial. An example of the former approach is the coating of asubstrate with preformed PAZAM using spin coating, dipping, flow of thegel under positive or negative pressure or techniques set forth in U.S.Prov. Pat. App. Ser. No. 61/753,833, which is incorporated herein byreference. Coating of an array of wells with preformed PAZAM isdemonstrated below in Example III. An example of applying liquid thatsubsequently forms a gel material is the coating of an array of wellswith silane free acrylamide and N-[5-(2-bromoacetyl) aminopentyl]acrylamide (BRAPA) in liquid form and allowing the reagents to form agel by polymerization on the surface. Coating of an array in this way isdemonstrated in Example I below and can use chemical reagents andprocedures as set forth in US Pat. App. Pub. No. 2011/0059865 A1, whichis incorporated herein by reference. In some embodiments, for example,when a well-containing substrate is dipped into a preformed gelmaterial, the gel material may fill the wells selectively and polishingmay not be necessary.

Analytes can be added to a gel material prior to contact with a solidsupport or afterward. Furthermore, analytes can be added to a gel (i.e.after the gel has formed from its precursor reagents) or analytes can beadded to a gel forming reagent solution (i.e. prior to gel formation).In some embodiments various analytes can be added prior to gel formationand others can be added after gel formation. In one example, primernucleic acids are added to a gel forming solution and the solution isthen allowed to form into a gel (e.g. by polymerization as occurs forSFA and PAZAM). The gel formation may occur on a solid support or thegel may be preformed and then coated onto a solid support. Either way,the primers will be attached to the gel that is present in concavefeatures such as wells. Target nucleic acids that are complementary tothe primers can then be added to the primer-containing gel such that thetarget nucleic acids become attached to the gel (via hybridization)after the gel material has been coated onto the solid support. Thehybridization of the target nucleic acids can optionally occur after apolishing step has been carried out (polishing is described in furtherdetail below). The preceding example, describes several instances wherenucleic acids (either functioning as primers or targets) are added to agel at different stages of the manufacture of a structured substrate.

In several embodiments, primer nucleic acids that are attached to a gel(or otherwise present in or on a gel) can be used for capture and/oramplification of template nucleic acids. The primers can be universalprimers that hybridize to a universal adapter sequence that is attachedto different target nucleic acids in a library (i.e. each target nucleicacid includes a target region that differs from other target nucleicacids in the library and several target nucleic acids in the libraryhave the same universal adapter sequence). In some embodiments, a targetnucleic acid can be attached to gel material, and primers (whether insolution or also attached to the gel) can be used to amplify theattached target nucleic acid (i.e. the target nucleic acid can serve asa template for amplification).

A method set forth herein can use any of a variety of amplificationtechniques. Exemplary techniques that can be used include, but are notlimited to, polymerase chain reaction (PCR), rolling circleamplification (RCA), multiple displacement amplification (MDA), orrandom prime amplification (RPA). In particular embodiments, one or moreprimers used for amplification can be attached to a gel material. In PCRembodiments, one or both of the primers used for amplification can beattached to a gel material. Formats that utilize two species of attachedprimer are often referred to as bridge amplification because doublestranded amplicons form a bridge-like structure between the two attachedprimers that flank the template sequence that has been copied. Exemplaryreagents and conditions that can be used for bridge amplification aredescribed, for example, in U.S. Pat. No. 5,641,658; U.S. Patent Publ.No. 2002/0055100; U.S. Pat. No. 7,115,400; U.S. Patent Publ. No.2004/0096853; U.S. Patent Publ. No. 2004/0002090; U.S. Patent Publ. No.2007/0128624; and U.S. Patent Publ. No. 2008/0009420, each of which isincorporated herein by reference. PCR amplification can also be carriedout with one of the amplification primers attached to a gel material andthe second primer in solution. An exemplary format that uses acombination of one solid phase-attached primer and a solution phaseprimer is emulsion PCR as described, for example, in Dressman et al.,Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), WO 05/010145, or U.S.Patent Publ. Nos. 2005/0130173 or 2005/0064460, each of which isincorporated herein by reference. Emulsion PCR is illustrative of theformat and it will be understood that for purposes of the methods setforth herein the use of an emulsion is optional and indeed for severalembodiments an emulsion is not used. Furthermore, primers need not beattached directly to solid supports as set forth in the ePCR referencesand can instead be attached to a gel material as set forth herein. Insome solid phase PCR or bridge amplification formats, a target nucleicacid can be attached to a gel material and used as a template foramplification.

RCA techniques can be modified for use in a method of the presentdisclosure. Exemplary components that can be used in an RCA reaction andprinciples by which RCA produces amplicons are described, for example,in Lizardi et al., Nat. Genet. 19:225-232 (1998) and US Pat. App. Pub.No. 2007/0099208 A1, each of which is incorporated herein by reference.Primers used for RCA can be in solution or attached to a gel material.

MDA techniques can be modified for use in a method of the presentdisclosure. Some basic principles and useful conditions for MDA aredescribed, for example, in Dean et al., Proc Natl. Acad. Sci. USA99:5261-66 (2002); Lage et al., Genome Research 13:294-307 (2003);Walker et al., Molecular Methods for Virus Detection, Academic Press,Inc., 1995; Walker et al., Nucl. Acids Res. 20:1691-96 (1992); U.S. Pat.Nos. 5,455,166; 5,130,238; and 6,214,587, each of which is incorporatedherein by reference. Primers used for MDA can be in solution or attachedto a gel material.

In particular embodiments a combination of the above-exemplifiedamplification techniques can be used. For example, RCA and MDA can beused in a combination wherein RCA is used to generate a concatamericamplicon in solution (e.g. using solution-phase primers). The ampliconcan then be used as a template for MDA using primers that are attachedto a gel material. In this example, amplicons produced after thecombined RCA and MDA steps will be attached to the gel material. Theamplicons will generally contain concatameric repeats of a targetnucleotide sequence.

Amplification techniques, such as those exemplified above, can be usedto produce gel-containing features having multiple copies of targetnucleic acids. An individual feature, such as a well, can have a clonalpopulation of nucleotide sequences in the form of a single moleculeconcatamer, such as those produced by RCA, or in the form of manynucleic acid molecules having the same sequence such as those producedby bridge PCR. Generally the nucleic acid(s) having several copies ofthe amplified target will be attached to the gel material.

For some applications, an individual gel-containing well (or otherconcave feature) can be predominantly populated with amplicons from afirst target nucleic acid and can also have a low level of contaminatingamplicons from a second target nucleic acid or from a spontaneousmutation occurring during amplification. An array can have one or moreamplification sites that have a sufficiently low level of contaminatingamplicons so as to have an unacceptable impact on a subsequent use ofthe array. For example, when the array is to be used in a detectionapplication, an acceptable level of contamination would be a level thatdoes not impact signal to noise or resolution of the detection techniquein an unacceptable way. Accordingly, apparent clonality will generallybe relevant to a particular use or application of an array made by themethods set forth herein. Exemplary levels of contamination that can beacceptable at an individual well or other feature for particularapplications include, but are not limited to, at most 0.1%, 0.5%, 1%,5%, 10% or 25% contaminating amplicons. An array can include one or morewells or other features having these exemplary levels of contaminatingamplicons. For example, up to 5%, 10%, 25%, 50%, 75%, or even 100% ofthe features in an array can have some contaminating amplicons.

A gel material that has been coated on the surface of a solid supportcan be covalently attached to the support. As set forth above, the stepof attaching an analyte, such as a nucleic acid, to gel material can becarried out at a variety of different stages in the manufacture of astructured substrate. Thus, a gel material can be attached to a solidsupport before or after attaching an analyte to the gel material.Attachment of gel material to a solid support can be carried out usingany useful chemistry including without limitation those set forth inU.S. Prov. Pat. App. Ser. No. 61/753,833, which is incorporated hereinby reference, or demonstrated in Example III below. It will beunderstood that covalent attachment of gel material to a solid supportis not necessary in all embodiments. Thus, the subsequent steps ofpolishing a gel-coated support or using a polished substrate can becarried out for a substrate having gel material that is optionally, butnot necessarily, covalently attached to concave features, such as wells.

A method set forth herein can include a step of removing gel materialfrom the surface of a solid support. Gel material that is coated on asolid support can be selectively removed from interstitial regions usingany of a variety of techniques. For example, gel material can be removedfrom a solid support having concave features and interstitial regions bya mechanical polishing technique. Mechanical polishing can be carriedout by applying abrasive forces to the surface of the solid support.Exemplary methods include abrasion with a slurry of beads, wiping with asheet or cloth, scraping or the like. It will be understood that beadsused for polishing or other uses set forth herein can be, but need notbe, spherical. Rather beads can have irregular shapes, polygonal shapes,ovoid shapes, elongated shapes, cylindrical shapes etc. The surface ofthe beads can be smooth or rough. Any of a variety of particles can beuseful as beads for the methods and compositions set forth herein. Oneexample of polishing includes using a lintless (cleanroom grade) wipecoated with a 3 μm silica bead slurry (10% w/v in water) to removeinterstitial gel. A polishing wheel/grinder can also be used with thisslurry. Mechanical polishing can also be achieved using a fluid jet orgas (e.g. air or inert gas such as Argon or Nitrogen) jet to remove gelfrom interstitial regions.

Polishing can involve chemical polishing such as hydrolysis orradical-based degradation of acrylamide (e.g. via exposure to benzoylperoxide or dilute hydrogen peroxide as described in Kurenkov, et al.,Russian Journal of Applied Chemistry, 75:1039-1050 (2002); Caulfield etal., Polym. 44:1331-1337 (2003); and Caulfield, et al., Chem. Rev.102:3067-3083 (2002). During polishing, chemicals can be provided in asolid, liquid, gas or plasma state. Accordingly, plasma polishing can beuseful in some embodiments.

Polishing can also involve a combination of chemical and mechanicalpolishing methods where a chemical slurry containing a colloidalsuspension of particles is used to mechanically exfoliate and thenchemically dissolve displaced portions of gel material from interstitialregions. Other methods to polish or clean the interstitial regionsinclude adhesive based techniques, for example, techniques wherein arigid, planar adhesive film with affinity to the gel material is coatedon the surface thereby making intimate contact (e.g. via chemicallinkage) with the gel material in interstitial regions. The mechanicalremoval/peeling of this adhesive film will result in the mechanicalremoval of the gel material from interstitial regions, while leaving gelmaterial in concave features.

In another example, thiophosphate-grafted SFA can be removed frominterstitial regions on a surface as follows. A water-dampened Whatmanwipe can be dabbed into Aluminium oxide (˜100 mg, 0.3 um) or steelbeads. Then, the formed slurry can be rubbed on the surface of a solidsupport, in small concentric circles, using even pressure. A cleanwater-wet Whatman wipe can then be used to remove the slurry on thesurface. The mechanical and chemical polishing methods exemplifiedherein for removing gel material from interstitial regions can also beused to inactivate gel material at interstitial regions, whether or notthe gel material is removed. For example, the gel material can beinactivated with respect to the ability to attach to analytes such asnucleic acids or with respect to the ability to support nucleic acidamplification.

A method of making an array can include the steps of (a) providing asolid support having a surface with a plurality of wells, the wellscontaining a gel material, the wells being separated from each other byinterstitial regions on the surface, the interstitial regionssegregating the gel material in each of the wells from the gel materialin other wells of the plurality; (b) delivering a library of targetnucleic acids to the wells of the solid support to produce an array ofwells that have a single species of target nucleic acid attached to thegel material in each well, wherein different wells in the array havedifferent target nucleic acid species from the library; and (c)amplifying the target nucleic acids attached to the gel material in thewells of the array to produce a clonal population of an individualtarget nucleic acid at each of the wells of the array.

In several embodiments, the structured substrates set forth hereinprovide the advantage of convenient delivery of multiple differentanalytes from a mixture to individualized locations on the substrate,thereby forming an array. The structured substrates facilitate selectivecapture of a single analyte at each individual gel-containing well (orother concave feature) from a mixture of analytes in contact with thesubstrate. The pattern of gel-containing wells (or other concavefeatures) on the structured substrate and the efficiency of loading canbe adjusted to obtain arrays having desired characteristics such asanalyte density and purity of each feature with respect to having asingle analyte species. For example, a higher density of wells can beused to obtain a higher density of analytes on the array and, converselya lower density of wells can be used to obtain a lower density ofanalytes on the array. Alternatively or additionally, the concentrationor amount of analyte in solution can be increased to obtain a higherdensity of analytes on the array or decreased to obtain a lower densityof analytes on the array. The average purity of analytes at eachgel-containing well (or other concave feature) can be adjusted byaltering properties of the substrate or conditions for delivery ofanalyte as set forth in further detail below and demonstrated in theExamples section.

In particular embodiments, the size or volume of the wells (or otherconcave features) can be adjusted to influence the purity of analytescaptured. For example, a well can have an area or volume of gel materialthat accommodates only a single analyte of a particular type such thatsteric exclusion prevents more than one analyte molecule from beingcaptured or seeding the well. Steric exclusion can be particularlyuseful for large analytes such as nucleic acids. More specifically,wells (or other concave features) can present a gel surface having anarea that is equivalent to or smaller than the diameter of the excludedvolume of the target nucleic acids that are to be seeded on thesubstrate. The excluded volume for a target nucleic acid and itsdiameter can be determined, for example, from the length of the targetnucleic acid. Methods for determining the excluded volume of nucleicacids and the diameter of the excluded volume are described, forexample, in U.S. Pat. No. 7,785,790; Rybenkov et al., Proc. Natl. Acad.Sci. U.S.A. 90: 5307-5311 (1993); Zimmerman et al., J. Mol. Biol.222:599-620 (1991); or Sobel et al., Biopolymers 31:1559-1564 (1991),each of which is incorporated herein by reference. Conditions for stericexclusion are set forth in U.S. Ser. No. 13/661,524 and U.S. Pat. No.7,785,790, each of which is incorporated herein by reference, and can bereadily used for structured substrates of the present disclosure.

It will be understood that in some embodiments, wells (or other concavefeatures) can present a gel surface having an area that is substantiallygreater than the diameter of the excluded volume of the target nucleicacids that are transported to the amplification sites. Thus, the areafor the features can be sufficiently large that steric exclusion doesnot occur.

In some embodiments, such as the steric exclusion embodiments set forthabove, a library of target nucleic acids can be delivered togel-containing wells (or other concave features) of a solid supportprior to initiation of an amplification process. For example, targetnucleic acids can be delivered to a structured substrate underconditions to seed the gel material in the substrate with the targetnucleic acids. The substrate can optionally be washed to remove targetnucleic acids that do not seed the gel as well as any other materialsthat are unwanted for subsequent processing or use of the substrate.Amplification can include one or more of the techniques set forthpreviously herein.

In alternative embodiments, a library of target nucleic acids can bedelivered to gel-containing wells (or other concave features) of a solidsupport and an amplification process can occur simultaneously with theseeding event. For example, seeding can occur under a regime thatexploits kinetic exclusion as described for example in U.S. Ser. No.61/715,478, which is incorporated herein by reference. Kinetic exclusioncan occur when a process occurs at a sufficiently rapid rate toeffectively exclude another event or process from occurring. In the caseof an array of gel-containing wells, the wells can be randomly seededwith target nucleic acids from a solution and copies of the targetnucleic acid can be generated in an amplification process to fill eachof the seeded sites to capacity. The seeding and amplification processescan proceed simultaneously under conditions where the amplification rateexceeds the seeding rate. As such, the relatively rapid rate at whichcopies are made at a site that has been seeded by a first target nucleicacid will effectively exclude a second nucleic acid from seeding thesite for amplification. Similarly, kinetic exclusion can exploit arelatively slow rate for making a first copy of a target nucleic acidvs. a relatively rapid rate for making subsequent copies of the targetnucleic acid or of the first copy. For example, kinetic exclusion canoccur due to a delay in the formation of a first copy of a targetnucleic acid that has seeded a gel-containing well (e.g. delayed or slowactivation) vs. the relatively rapid rate at which subsequent copies aremade to fill the site. In this example, an individual gel-containingwell may have been seeded with several different target nucleic acids(e.g. several target nucleic acids can be present at each site prior toamplification). However, first copy formation for any given targetnucleic acid can be activated randomly such that the average rate offirst copy formation is relatively slow compared to the rate at whichsubsequent copies are generated. In this case, although an individualgel-containing well may have been seeded with several different targetnucleic acids, kinetic exclusion will allow only one of those targetnucleic acids to be amplified. Generally, a gel-containing well (orother concave feature) can serve as a site for amplification and arrayformation in a method set forth in U.S. Ser. No. 61/715,478, which isincorporated herein by reference.

As an alternative to delivery of multiple different analytes from amixture to individual gel-containing concave features, analytes can bediscretely delivered to individual features from pure stocks. Similarly,analytes can be synthesized at individual features by discrete deliveryof synthetic building blocks (e.g. nucleotide precursors can besequentially delivered to synthesize nucleic acids). Exemplary methodsfor delivery of pure analytes or building blocks for synthesizinganalytes in situ include, but are not limited to, ink jet array spottingand photolithographic array synthesis. Useful photolithographic methodsinclude those used commercially by Affymetrix (Santa Clara, Calif.) tomanufacture GeneChip® microarrays or described in U.S. Pat. Nos.5,324,633; 5,744,305; 5,624,711; 6,022,963; 6,291,183; and 6,416,949,each of which is hereby incorporated by reference. Also useful areinkjet spotting techniques such as those commercialized by Agilent(Santa Clara, Calif.) for printing SurePrint™ arrays or described inU.S. Pat. Nos. 6,337,393; 6,419,883; 6,420,180 or 6,689,319, each ofwhich is incorporated herein by reference. Such methods can be readilymodified to direct delivery to gel-containing features of the presentdisclosure.

The gel material in a particular concave feature need not contain only asingle species of analyte. Rather, in some embodiments a concave featurecan contain several different species of analyte in the gel therein. Anexample is demonstrated by the bulls-eye fiducial markers in FIG. 5. Thefiducial markers include two ‘bright’ ring shaped channels, each of thetwo channels contains gel material, and the gel material in each brightchannel is attached to a plurality of different nucleic acid colonies.The nucleic acid colonies in the bright channels were formed by seedingof each ring with several different species of target nucleic acid thatfunctioned as templates in an amplification procedure. The fiducialmarker also includes two ‘dark’ ring shaped regions. The dark rings areformed by interstitial surface patterns. The exemplary bulls-eye isformed by alternating dark and bright rings in a concentric pattern. Inthe example of FIG. 5 the structured substrate also includesgel-containing wells that each generally contains a clonal populationderived from a single nucleic acid target. The wells occur in a ringshaped band between the bright and dark rings. Thus, the fiducials havean alternating pattern of interstitial ring, well-containing band andchannel ring. The same amplification procedure was used tosimultaneously grow the clonal nucleic acid colonies in the wells andthe mixed population in the fiducial marker (see Example III, below).Other examples of fiducials having alternating patterns of rings areshown in FIG. 3B and FIG. 3C.

This disclosure further provides a method of detecting analytes. Themethod can include the steps of (a) providing a solid support having aplanar surface, wherein the planar surface is interrupted by one or moreconcave features, wherein the concave features contain gel material,wherein the one or more concave features are bordered by one or moreinterstitial regions on the planar surface, the interstitial regionsbeing substantially devoid of the gel material, and wherein the gelmaterial is attached to or contains target analytes; (b) contacting thesolid support with probes under conditions wherein the target analytesinteract specifically with the probes; and (c) detecting the solidsupport to distinguish at least a subset of the target analytes thatinteract with one or more of the probes.

In particular embodiments nucleic acids are the analytes that aredetected and the concave features are wells. For example a method ofdetecting nucleic acids can include the steps of (a) providing a solidsupport having a surface and a library of nucleic acids, the surfacehaving a plurality of wells, the wells containing a gel material, thewells being separated from each other by interstitial regions on thesurface, the interstitial regions segregating the gel material in eachof the wells from the gel material in other wells of the plurality, asingle species of the target nucleic acids of the library being attachedto the gel material in each of the wells; (b) contacting the solidsupport with at least one probe that binds to the target nucleic acids;and (c) detecting the solid support to distinguish the wells having atarget nucleic acid species that binds to the at least one probe.

Structured substrates of the present disclosure that contain nucleicacid arrays can be used for any of a variety of purposes. A particularlydesirable use for the nucleic acids is to serve as capture probes thathybridize to target nucleic acids having complementary sequences. Thetarget nucleic acids once hybridized to the capture probes can bedetected, for example, via a label recruited to the capture probe.Methods for detection of target nucleic acids via hybridization tocapture probes are known in the art and include, for example, thosedescribed in U.S. Pat. No. 7,582,420; 6,890,741; 6,913,884 or 6,355,431or US Pat. App. Pub. Nos. 2005/0053980 A1; 2009/0186349 A1 or2005/0181440 A1, each of which is incorporated herein by reference. Forexample, a label can be recruited to a capture probe by virtue ofhybridization of the capture probe to a target probe that bears thelabel. In another example, a label can be recruited to a capture probeby hybridizing a target probe to the capture probe such that the captureprobe can be extended by ligation to a labeled oligonucleotide (e.g. vialigase activity) or by addition of a labeled nucleotide (e.g. viapolymerase activity).

A nucleic acid array can also be used in a sequencing procedure, such asa sequencing-by-synthesis (SBS) technique. Briefly, SBS can be initiatedby contacting the target nucleic acids with one or more labelednucleotides, DNA polymerase, etc. Those features where a primer isextended using the target nucleic acid as template will incorporate alabeled nucleotide that can be detected. Optionally, the labelednucleotides can further include a reversible termination property thatterminates further primer extension once a nucleotide has been added toa primer. For example, a nucleotide analog having a reversibleterminator moiety can be added to a primer such that subsequentextension cannot occur until a deblocking agent is delivered to removethe moiety. Thus, for embodiments that use reversible termination, adeblocking reagent can be delivered to the flow cell (before or afterdetection occurs). Washes can be carried out between the variousdelivery steps. The cycle can then be repeated n times to extend theprimer by n nucleotides, thereby detecting a sequence of length n.Exemplary SBS procedures, fluidic systems and detection platforms thatcan be readily adapted for use with an array produced by the methods ofthe present disclosure are described, for example, in Bentley et al.,Nature 456:53-59 (2008), WO 04/018497; WO 91/06678; WO 07/123744; U.S.Pat. Nos. 7,057,026; 7,329,492; 7,211,414; 7,315,019 or 7,405,281, andUS Pat. App. Pub. No. 2008/0108082 A1, each of which is incorporatedherein by reference.

Other sequencing procedures that use cyclic reactions can be used, suchas pyrosequencing. Pyrosequencing detects the release of inorganicpyrophosphate (PPi) as particular nucleotides are incorporated into anascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi etal. Science 281(5375), 363 (1998); U.S. Pat. Nos. 6,210,891; 6,258,568and 6,274,320, each of which is incorporated herein by reference). Inpyrosequencing, released PPi can be detected by being converted toadenosine triphosphate (ATP) by ATP sulfurylase, and the resulting ATPcan be detected via luciferase-produced photons. Thus, the sequencingreaction can be monitored via a luminescence detection system.Excitation radiation sources used for fluorescence based detectionsystems are not necessary for pyrosequencing procedures. Useful fluidicsystems, detectors and procedures that can be used for application ofpyrosequencing to arrays of the present disclosure are described, forexample, in WIPO Pat. App. Ser. No. PCT/US11/57111, US Pat. App. Pub.No. 2005/0191698 A1, U.S. Pat. Nos. 7,595,883, and 7,244,559, each ofwhich is incorporated herein by reference.

Sequencing-by-ligation reactions are also useful including, for example,those described in Shendure et al. Science 309:1728-1732 (2005); U.S.Pat. Nos. 5,599,675; and 5,750,341, each of which is incorporated hereinby reference. Some embodiments can include sequencing-by-hybridizationprocedures as described, for example, in Bains et al., Journal ofTheoretical Biology 135(3), 303-7 (1988); Drmanac et al., NatureBiotechnology 16, 54-58 (1998); Fodor et al., Science 251(4995), 767-773(1995); and WO 1989/10977, each of which is incorporated herein byreference. In both sequencing-by-ligation andsequencing-by-hybridization procedures, nucleic acids that are presentin gel-containing wells (or other concave features) are subjected torepeated cycles of oligonucleotide delivery and detection. Fluidicsystems for SBS methods as set forth herein, or in references citedherein, can be readily adapted for delivery of reagents forsequencing-by-ligation or sequencing-by-hybridization procedures.Typically, the oligonucleotides are fluorescently labeled and can bedetected using fluorescence detectors similar to those described withregard to SBS procedures herein or in references cited herein.

Some embodiments can utilize methods involving the real-time monitoringof DNA polymerase activity. For example, nucleotide incorporations canbe detected through fluorescence resonance energy transfer (FRET)interactions between a fluorophore-bearing polymerase andγ-phosphate-labeled nucleotides, or with zeromode waveguides. Techniquesand reagents for FRET-based sequencing are described, for example, inLevene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett.33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci. USA 105,1176-1181 (2008), the disclosures of which are incorporated herein byreference.

Some SBS embodiments include detection of a proton released uponincorporation of a nucleotide into an extension product. For example,sequencing based on detection of released protons can use an electricaldetector and associated techniques that are commercially available fromIon Torrent (Guilford, Conn., a Life Technologies subsidiary) orsequencing methods and systems described in US Pat. App. Pub. Nos.2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1; or 2010/0282617 A1,each of which is incorporated herein by reference. In particularembodiments, the electrical detectors that are used to detect thereleased protons can be modified to include wells and the wells cancontain gel material as set forth herein.

Another useful application for an array of the present disclosure isgene expression analysis. Gene expression can be detected or quantifiedusing RNA sequencing techniques, such as those, referred to as digitalRNA sequencing. RNA sequencing techniques can be carried out usingsequencing methodologies known in the art such as those set forth above.Gene expression can also be detected or quantified using hybridizationtechniques carried out by direct hybridization to an array or using amultiplex assay, the products of which are detected on an array. Anarray of the present disclosure can also be used to determine genotypesfor a genomic DNA sample from one or more individual. Exemplary methodsfor array-based expression and genotyping analysis that can be carriedout on an array of the present disclosure are described in U.S. Pat. No.7,582,420; 6,890,741; 6,913,884 or 6,355,431 or US Pat. App. Pub. Nos.2005/0053980 A1; 2009/0186349 A1 or 2005/0181440 A1, each of which isincorporated herein by reference.

Several applications for arrays of the present disclosure have beenexemplified above in the context of ensemble detection, wherein multiplecopies of a target nucleic acid are present at each feature and aredetected together. In alternative embodiments, a single nucleic acid,whether a target nucleic acid or amplicon thereof, can be detected ateach feature. For example, a gel-containing well (or other concavefeature) can be configured to contain a single nucleic acid moleculehaving a target nucleotide sequence that is to be detected. Any of avariety of single molecule detection techniques can be used including,for example, modifications of the ensemble detection techniques setforth above to detect the sites at increased resolution or using moresensitive labels. Other examples of single molecule detection methodsthat can be used are set forth in US Pat. App. Pub. No. 2011/0312529 A1;U.S. Ser. No. 61/578,684; and U.S. Ser. No. 61/540,714, each of which isincorporated herein by reference.

It will be understood that a gel-containing substrate of the presentdisclosure, for example, having been produced by a method set forthherein, need not be used for a detection method. Rather, the structuredsubstrate can be used to store a nucleic acid library. Accordingly, thestructured substrate can be stored in a state that preserves the nucleicacids therein. For example, a substrate having gel-containing wells thatare attached to nucleic acids can be stored in a desiccated state,frozen state (e.g. in liquid nitrogen), or in a solution that isprotective of nucleic acids. Alternatively or additionally, thestructured substrate can be used to replicate a nucleic acid library.For example, a substrate having gel-containing wells that are attachedto nucleic acids can be used to create replicate amplicons from one ormore of the wells on the array.

The following examples are intended to illustrate but not limit thepresent invention.

Example I Multiwell Substrates Coated with Silane Free Acrylamide

This example demonstrates coating of nanowell substrates with silanefree acrylamide (SFA), followed by grafting with thiophosphate primersand performing a hybridization of the gel-grafted primers tocomplimentary fluorescent oligonucleotide to confirm the success of thefunctionalization approach.

Chip substrates normally used for manufacture of BeadChips were obtainedfrom Illumina (San Diego, Calif.). The chips were made of silicon orZeonor (Zeon Corp., Tokyo, Japan) having 0.5 μm wells arranged in ahexagonal pattern having a pitch of 1.5 μm, but the wells did notcontain beads. The chips were patterned with gel pads as set forth belowand diagrammed in FIG. 1.

The chips were encased in a gasket sealed chamber and oxygen was removedby displacement with fluid reagents for formation of SFA. SFA waspolymerized on the chips in the chamber. The reagents for formation ofSFA and conditions for polymerization were otherwise as described in USPat. App. Pub. No. 2011/0059865 A1, which is incorporated herein byreference. The sealed chamber was used to place the polymerizing mix indirect contact with the chip and to ensure complete elimination of airsince free radical polymerization of SFA is an air-sensitive process.After polymerization, primers were grafted to the SFA polymer in thesealed chamber as set forth in US Pat. App. Pub. No. 2011/0059865 A1 andas follows. A solution containing the primers was drawn across thepolymer coated surface of the BeadChip and the mix was then incubatedfor 1.25 h at 65° C. (the entire sealed assembly was placed in a largeoven).

This approach gave uniformly coated substrates. A sample image is shownin FIG. 2, Panel A. In order to create discrete polymer regions, the‘excess’ polymer situated between the wells on the substrate was removedusing a mechanical polishing technique using a slurry of aluminum oxidenanoparticles (300 nm diameter) in DI water. A 10 wt. % slurry of 3micron silica particles (Kisker Biotech GmbH, Steinfurt, Germany) canalso be used. The surface was manually scrubbed with the nanoparticleslurry using a lint-free optical tissue. After washing to remove theslurry and polymer debris, a solution of fluorescently labeled probeswas hybridized to the chip. Images captured using a fluorescencemicroscope showed that this approach was capable of yielding polymerfeatures with clean interstitial regions (FIG. 2, Panels B through C).

These results indicated that the fluorescent intensities at thegel-filled wells were spatially discrete in contrast to the absence ofsignal from the interstitial regions. The results also demonstrated thatgel patterning can be achieved using non-covalently attached gelmaterial on a substrate having nanofabricated wells.

Example II Fabrication of a Substrate Having Gel-Containing Nanowells

Multiple techniques can be used to fabricate structured arrays that cansubsequently be loaded with gel material.

The process can start with a blank substrate/wafer and a pattern isintroduced into the substrate via micro- or nano-fabrication techniques.The material of the substrate/wafer can be conventional silicon, glass,plastic, COC or any of a variety of materials that can be structured.Exemplary techniques for introducing the patterning into the substrateinclude photolithography, nanoimprint lithography, embossing of thestructures into a plastic/COC based material and injection molding of aplastic or COC into a master mold that has the structures patterned intoit. Photolithography based approaches will typically involve use of aphotoresist that is patterned with a stepper or mask aligner, exposedwith radiation which transfers the pattern present on areticle/photomask into the photoresist, and then the resist is developedto yield a structured film (photoresist) on top of the substrate. Thestructured resist is potentially the final substrate which can be usedfor subsequent gel coating or the pattern in the resist can betransferred into the substrate via follow on processing. The follow onprocess steps will typically include reactive ion etching (plasma basedetching) or a wet etch (chemically based) process. If the pattern istransferred into the substrate, the patterned photoresist issubsequently removed to yield the patterned substrate for subsequent gelcoating. It may be desirable to use a sacrificial film of a materialsuch as Chrome or Titanium (a metal) under the photoresist, and firsttransfer the pattern in the photoresist to the metal film and then usethat film as a hard mask by which the pattern is transferred into thesubstrate. Following pattern transfer into the substrate, the films areremoved and therefore considered sacrificial to the fabrication process.If nanoimprint lithography is used, the imprinted photoresist can be asacrificial material and similarly be used as an intermediate tool totransfer the patterned resist into the substrate or a variation of theresist can be used such that the imprinted resist serves as the input toa subsequent coating step. An example of a resist that would remainfollowing patterning would be a Sol-Gel based material.

A diagrammatic representation of how a structured substrate may befabricated is shown in FIG. 3 and described below. Images of patternedsubstrates are shown at various magnification levels in FIG. 3B and FIG.3C.

Creation of chemically specific gel pads on a sequencingsubstrate/flowcell can involve one or more of the nanofabricationtechniques set forth previously in this Example. The process mayoptionally then include one or more chemical processing steps, such assilanization to allow subsequent linking of a gel polymer to thesubstrate via the silane. Then chemical/mechanical polishing (CMP) isused to remove all interstitial polymer on the surface of the substrate.The polishing process will remove material in a top down fashion andsince the structured features in the substrate are effectively offsetfrom the plain of the interstitial regions of the array, the polishingwill remove the polymer from the interstitial prior to removing thestructured features. If the polishing process is stopped after theoptimum time, the structures will retain the polymer coating and theinterstitial regions will be void of the polymer. The patterned gel padsubstrate is then grafted with primers, target nucleic acids are seededat the gel pads and the target nucleic acids are used as templates forcreation of nucleic acid clusters at the gel pads.

Example III Multiwell Substrates Coated with PAZAM

This example shows the fabrication of an array of gel-containing wells,amplification of nucleic acid clusters in the wells and sequencing ofthe nucleic acids in the clusters.

Substrates were fabricated as follows. A nanowell substrate (400 nmdiameter 1.5 μm pitch, 300 nm depth well) was fabricated usingnanoimprint lithography. An amino silane (APTES or APTMS)monolayer/multilayer was deposited on the entire surface of thesubstrate using chemical vapor deposition. Next, a 1× phosphate bufferedsaline (pH 7.4) solution of acrylic acid N-hydroxysuccinimide ester(Aldrich PN 8060) at 100 mM concentration was reacted with the aminosilane surface by adding 1 ml of the NHS acrylate solution to thesurface, covering it with a thin glass coverslip and allowing thereaction to proceed for 1 hour at room temperature. A polymer (PAZAM)was then applied to the surface by spin coating 500 μl of a 2 wt. %PAZAM solution in water onto the newly formed acrylamide functionalizedsurface. PAZAM was synthesized as described in U.S. Prov. Pat. App. Ser.No. 61/753,833, which is incorporated herein by reference. Subsequentheating of the PAZAM-coated substrate at 60° C. for 1 hour resulted in acovalent linkage between the polymer and surface. The interstitialcovalently linked polymer was removed by polishing the surface with 10wt. % of 3 μm SiO₂ micro particle slurry in water. A Janeway surface(acryloyl chloride with DIPEA in MeCN) can be used in place of the aminosilane coated surface in the above procedure.

The patterned polymer substrate was then grafted with primers asdescribed in U.S. Prov. Pat. App. Ser. No. 61/753,833, which isincorporated herein by reference. Next, dye-labeled (Cy5) reversecompliments of the grafted primers were exposed to the surface in a1×PBS buffer solution at a compliment concentration of 20 μM, and thenthe surface was washed with 50 ml of 1PBS buffer applied with a squirtbottle. The labeled complements on the substrate were imaged with a FLA9500Typhoon Imager set at the Cy5 scan channel and using a PMT settingof 450. The labeled compliments on the substrate were also imaged with ahigh resolution microscope, showing the patterning or polymer/primerswith no interstitial polymer/primers remaining (FIG. 4). The substratewas then seeded with phiX DNA, and clusters grown as described in U.S.Ser. No. 61/715,478, which is incorporated herein by reference.

A flow cell containing the cluster-containing substrate was sequenced ona HiSeq 2000 (Illumina, Inc., San Diego, Calif.). An algorithm toextract the locations of the patterned sequencing clusters (rigidregistration) was employed, successfully giving high quality sequencingmetrics (FIG. 5 and FIG. 6). Sequencing results showed that theoccupancy vs. clonality was surprisingly higher than expected for astandard Poisson distribution. Specifically, the average occupancy vs.clonality measure for the sequencing run, as indicated by the “x” inFIG. 7, is above the boundaries of the Poisson curve and approachesclosely to the line for the ideal clonal fraction).

Throughout this application various publications, patents or patentapplications have been referenced. The disclosures of these publicationsin their entireties are hereby incorporated by reference in thisapplication in order to more fully describe the state of the art towhich this invention pertains.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the invention. Accordingly, the inventionis limited only by the claims.

What is claimed is:
 1. An array comprising: a substrate; and a patternedsol-gel layer disposed on the substrate; wherein the patterned sol-gellayer comprises an array of a plurality of wells.
 2. The array of claim1, wherein the patterned sol-gel layer is a sintered sol-gel layer. 3.The array of claim 1, further comprising a gel material disposed in thewells, wherein the gel material is adapted to promote capture and/oramplification of target nucleic acids.
 4. The array of claim 3, whereinthe gel material comprises apoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM)polymer.
 5. The array of claim 3, wherein the gel material comprises asilane free acrylamide.
 6. The array of claim 3, wherein the gelmaterial is adapted such that, when a solution of the target nucleicacids contacts the gel material, a single species of the target nucleicacids attaches to the gel material in each well, optionally via primernucleic acids.
 7. The array of claim 1, wherein the plurality of wellsare arranged in one or more sub-patterns.
 8. The array of claim 1,wherein each of the plurality of wells has a depth selected from a groupconsisting of 1×10³ μm or less, 100 μm or less, 10 μm or less, 1 μm orless, and 0.1 μm or less.
 9. A flow cell comprising the array ofclaim
 1. 10. A patterned surface comprising: a substrate; and apatterned layer disposed on the substrate; wherein the patterned layercomprises an array of a plurality of wells, the plurality of wellscomprising analyte features and fiducial features.
 11. The patternedsurface of claim 10, wherein the plurality of wells are arranged in ahexagonal pattern.
 12. The patterned surface of claim 10, wherein thefiducial features comprise one or more sets of concentric rings.
 13. Thepatterned surface of claim 10, wherein the fiducial features comprise afirst subregion and a second subregion rotated with respect to the firstsubregion.
 14. The patterned surface of claim 10, wherein the fiducialfeatures comprise one or more sets of grids.
 15. The patterned surfaceof claim 10, wherein the patterned layer further comprises interstitialregions separating the plurality of wells.
 16. The patterned surface ofclaim 15, further comprising a gel material disposed in the wells,wherein the interstitial regions are devoid of the gel material.
 17. Apatterned surface comprising: a substrate; and a patterned layerdisposed on the substrate; wherein the patterned layer comprises aplurality of analyte features and fiducial features, wherein the analytefeatures and the fiducial features contain a polymer, and wherein primernucleic acids are attached to the polymer.
 18. The patterned surface ofclaim 17, further comprising one or more colonies of target nucleicacids captured by the primer nucleic acids for each of the plurality ofanalyte features and fiducial features.
 19. The patterned surface ofclaim 18, wherein the target nucleic acids captured by the primernucleic acids of the fiducial features comprise a fluorescent label.